Inhibitors of 2 of the 3 retroviral enzymes (reverse transcriptase and protease) are efficient anti-HIV drugs. Integrase (the 3rd retroviral enzyme) is also a rationale drug target because it is required for viral replication and does not have a known cellular equivalent. Two integrase inhibitors have recently started clinical trials. To extend the target range for development of anti-retroviral agents, we are focusing on HIV-1 integrase inhibitors using recombinant enzyme and short oligonucleotides corresponding to the proviral ends (LTR's). Integrase is a rationale target for drug development because it is essential for viral replication. It is encoded by HIV and does not have a cellular equivalent. Our laboratory has pioneered the integrase inhibitors research field, discovered several families of lead inhibitors and patented some with the aim of therapeutic development. Recently, the first class of selective HIV-1 integrase inhibitors with demonstrated antiviral activity related to integrase inhibition has been introduced in clinical trials. We are investigating these diketo acid (DKA) derivatives in collaboration with Dr. Terrence Burke (Laboratory of Medicinal Chemistry, CCR, NCI) and Dr. Vinay Pathak (Antiviral Drug Resistance Program, CCR, NCI). Our goals are to elucidate the molecular pharmacology of the DKA, and more specifically their binding site in the integrase-DNA complex. We are performing structure-activity relationship and collaborating with David Davies' crystallography group (NIDDK). We found that azido derivatives of diketo acids are potent and selective anti-integrase inhibitors and are antiviral. A patent application has been filed for our derivatives. We recently found in collaboration with Dr. Peter Roller (Laboratory of Medicinal Chemistry, CCR, NCI) that cationic peptides derived from indolicidin are integrase inhibitors. Their mechanism of action is novel as they bind to DNA, and thereby interfere with the formation of competent integrase-DNA complexes. In parallel, we are studying the molecular interactions between integrase and its DNA substrate using site-directed mutagenesis and enzyme-DNA crosslinking assays.Our recent studies supported by an IATAP grant demonstrate that integrase inhibitors can be screened using a novel high-throughput assay based on electrochemiluminescence (BioVeris). Using recombinant HIV-1 integrase, we have now screened the 3161 compounds from the three NCI Developmental Therapeutics Program (DTP) chemical libraries. A total of 123 compounds are inhibitory at 10 M (3.9% hit ratio). These 123 lead compounds are being examined in gel-based assays to evaluate their potency and selectivity for particular steps of the integrase reactions. The compounds are prioritized in collaboration with our colleagues from the NCI-CCR Laboratory of Medicinal Chemistry, the HIV Drug Resistance Program and the HIV and AIDS Malignancy Branch. Highest priority compounds are examined for mechanism of action, antiviral activity and structure-activity relationship with the aim of optimizing the drugs for medicinal development. We just filed a patent application for tropolones as anti-integrase inhibitors and anti-HIV drugs. We also discovered several tetracyclines active against integrase and with anti-HIV activity (recent patent from The Johns Hopkins University). Additional compounds with structures differing from know inhibitors are being pursued with the aim of providing novel anti-integrase drugs that can be patented and developed as new therapeutics against HIV and AIDS. The novel compounds will also be added to our pharmacophore collection and used to select compounds to be screened from other chemical libraries (ChemNavigator and NIH Roadmap initiative).